Analysis of Creatine and Creatinine in Dietary Supplements Using HILIC-MS

Significance of the topic

The reliable measurement of creatine and its degradation product creatinine in dietary supplements is important for product quality, label compliance, and consumer safety. Creatine supplements are widely used for athletic performance and emerging therapeutic and cognitive applications; manufacturers must therefore control production and storage to minimize non-enzymatic conversion of creatine to creatinine. Analytical approaches that retain and selectively detect these small, highly polar molecules while remaining compatible with mass spectrometric detection are needed for routine quality control and stability assessment.

Objectives and study overview

This application study developed and evaluated a hydrophilic interaction liquid chromatography–mass spectrometry (HILIC‑MS) method to quantify creatine and creatinine in commercial dietary supplements. Goals included establishing chromatographic conditions that yield strong retention and good peak shape for both analytes, achieving a linear calibration across a practical concentration range, verifying label-stated creatine content in three consumer products, and assessing creatinine levels as an indicator of degradation or formulation-dependent conversion.

Methodology

• Calibration and standards: Creatine and creatinine stock solutions prepared at 2 mg/mL in water were combined and diluted with 95:5 acetonitrile:water to produce standards at 1, 10, 50, 100, and 200 µg/mL. Bracketed triplicate injections before and after samples provided n = 6 per level for calibration.
• Sample preparation: Two powdered supplements and one tablet formulation were tested. Powder samples were dissolved to provide an equivalent of 5 g creatine in 500 mL water and then diluted to a nominal 10 µg/mL (final solvent 95:5 acetonitrile:water). The tablet was extracted with 90:10 acetonitrile:water, vortexed, sonicated, centrifuged, filtered (0.2 µm nylon) and diluted to 10 µg/mL.
• Chromatography: Screening compared three HILIC stationary phases (BEH Amide, BEH HILIC unbonded, and BEH Z-HILIC zwitterionic) using a high-organic starting mobile phase. The selected method used an ACQUITY Premier BEH HILIC Column (2.1 × 50 mm, 1.7 µm), column temperature 30 °C, flow 0.4 mL/min, injection 1 µL, and sample temperature 10 °C. Mobile phases were water (A), acetonitrile (B) and a 200 mM ammonium formate solution (D, pH 3.0) for buffering.
• Detection and quantitation: Mass detection was performed with an ACQUITY QDa mass detector using selected/ extracted ion monitoring. Calibration covered 1–200 µg/mL and was evaluated for linearity and precision using bracketed injections.

Used instrumentation

• ACQUITY UPLC H-Class Plus System with Column Manager and ACQUITY UPLC PDA Detector
• ACQUITY QDa Mass Detector for MS detection
• Columns evaluated: ACQUITY Premier BEH Amide (2.1 × 50 mm, 1.7 µm), ACQUITY Premier BEH HILIC (2.1 × 50 mm, 1.7 µm), Atlantis Premier BEH Z‑HILIC (2.1 × 50 mm, 1.7 µm)
• QuanRecovery vials with MaxPeak High Performance Surfaces, 0.2 µm nylon filters
• Data system: Empower Chromatography Data System (CDS)

Main results and discussion

• Column selection: The unbonded BEH HILIC column provided the best combination of signal intensity and peak shape for creatine and creatinine. Differences between stationary phases were attributed to multimodal retention contributions: partitioning into an adsorbed water layer, ionic interactions (notably cation-exchange for positively charged analytes under the chosen conditions), and hydrogen bonding. BEH Amide and Z‑HILIC showed poorer creatine peak shape and lower signal, likely because multi‑modal interactions broadened peaks.
• Calibration and linearity: Both analytes displayed good linear response across 1–200 µg/mL with R2 = 0.998 for creatine and R2 = 0.995 for creatinine using bracketed triplicate injections (n = 6 per level).
• Quantification in consumer samples: Calculated creatine concentrations in the three products agreed with the nominal prepared concentration within 8%, demonstrating the method’s quantitative accuracy and minimal artifactual degradation during analysis.
• Creatinine findings: Diluted sample analyses showed very low creatinine levels; Powder A showed no detectable creatinine, while Powder B and the Tablet contained measurable creatinine. Analyses of undiluted stock solutions revealed higher creatinine levels in some samples that fell within the method’s dynamic range, suggesting that dilution strategy and matrix effects influence detectability.
• Matrix effects and separations: Additional ingredients (e.g., amino acids, flavorants, malic acid) were detected and generally well resolved from creatine/creatinine. Malic acid partially co-eluted with creatine but ionized preferentially in ESI‑ mode whereas creatine ionized in ESI+; this orthogonality allowed clear identification. The data indicate formulation-dependent variability in creatinine content, possibly due to manufacturing conditions, storage, or the presence of even weak acids that can catalyze conversion to creatinine.
• Method advantages: HILIC enabled strong retention of polar analytes without non-volatile ion-pair reagents, supporting MS detection and improved selectivity and sensitivity versus ion-pairing reversed-phase approaches.

Benefits and practical applications

• Provides a sensitive, MS‑compatible assay suitable for routine QC of creatine supplements to verify label claims and monitor degradation.
• Retains small polar analytes effectively, enabling separation of creatine, creatinine, and co-formulants such as amino acids and organic acids.
• Avoids non-volatile buffers and ion-pair reagents, simplifying maintenance and enabling mass-based selectivity for identification and quantitation.
• Capable of screening products for process- or storage-induced creatinine formation, informing manufacturing controls and shelf‑life assessments.

Future trends and opportunities

• Further refinement of stationary-phase chemistry and a deeper mechanistic understanding of HILIC retention (partitioning vs ionic interactions) will improve method robustness and predictability for charged polar analytes.
• Integration of isotopically labeled internal standards would strengthen quantitation and correct for matrix effects and ionization variability.
• High-resolution mass spectrometry (HRMS) or hybrid workflows could expand capabilities to non-targeted screening for impurities, degradation products, and contaminants.
• Automation and higher-throughput sample preparation strategies would facilitate routine QC in manufacturing and regulatory contexts.
• Development of validated stability-indicating HILIC-MS assays for regulatory submissions and shelf-life determination of dietary supplements.

Conclusion

The presented HILIC‑MS approach using an ACQUITY Premier BEH HILIC stationary phase and an ACQUITY QDa detector provides robust retention, good peak shape, and linear quantitation for creatine and creatinine across 1–200 µg/mL. The method accurately verified creatine content of three consumer products and detected variable creatinine levels consistent with formulation and/or processing differences. By avoiding non‑volatile reagents and enabling MS detection, this workflow is well suited for routine quality control and characterization of creatine supplements.

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